Triazine derivatives for the treatment of conditions associated with nicotinamide adenine dinucleotide phosphate oxidase

Abstract

A compound of formula (I) for use in the treatment of a condition or disorder associated with nicotinamide adenine dinucleotide phosphate oxidase.

Claims

1. A method of inhibiting NOX4 in a mammal, wherein the inhibition of NOX4 is used to treat a disorder or condition selected from the group consisting of endocrine disorders, cardiovascular disorders, respiratory disorders, metabolism disorders, skin disorders, bone disorders, neuroinflammatory and/or neurodegenerative disorders, kidney diseases, reproduction disorders, diseases affecting the eye and/or the lens and/or conditions affecting the inner ear, inflammatory disorders, liver diseases, pain, cancers, lung cancer, allergic disorders, traumatisms, septic, hemorrhagic and anaphylactic shock, diseases or disorders of the gastrointestinal system, angiogenesis, angiogenesis-dependent conditions, lung infections, acute lung injury, pulmonary arterial hypertension, obstructive lung disorders, and fibrotic lung disease, the method comprising administering, to a mammal in need thereof, a compound of formula (I) ##STR00020## wherein each R.sup.1, R.sup.2 and R.sup.3 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen; each R.sup.4 is independently selected from halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen; each R.sup.5 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen; each R.sup.6 and R.sup.7 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen; X is NH, CH.sub.2 or C(O); Y is NH; CH.sub.2 or C(O); m is an integer of from 0 to 5; n is an integer of from 0 to 5; p is an integer of from 0 to 4; and q is an integer of from 0 to 3; or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein each R.sup.1 is independently selected from halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

3. The method according to claim 1, wherein each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, and R.sup.5S(CH.sub.2).sub.q.

4. The method according to claim 2, wherein each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, and R.sup.5S(CH.sub.2).sub.q.

5. The method according to claim 1, wherein R.sup.3 is R.sup.6R.sup.7N(CH.sub.2).sub.q.

6. The method according to claim 2, wherein R.sup.3 is R.sup.6R.sup.7N(CH.sub.2).sub.q.

7. The method according to claim 3, wherein R.sup.3 is R.sup.6R.sup.7N(CH.sub.2).sub.q.

8. The method according to claim 4, wherein R.sup.3 is R.sup.6R.sup.7N(CH.sub.2).sub.q.

9. The method according to claim 1, wherein both R.sup.6 and R.sup.7 are H.

10. The method according to claim 5, wherein both R.sup.6 and R.sup.7 are H.

11. The method according to claim 1, wherein m is an integer of from 0 to 2.

12. The method according to claim 1, wherein n is 0 or 1.

13. The method according to claim 1, wherein p is 0.

14. The method according to claim 1, wherein X is NH.

15. The method according to claim 1, wherein Y is CH.sub.2.

16. The method according to claim 14, wherein Y is CH.sub.2.

17. The method according to claim 1, wherein each R.sup.1 is independently selected from halogen and C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen; each R.sup.2 is independently selected from halogen and R.sup.5O; R.sup.3 is NH.sub.2; R.sup.5 is C1-C6 alkyl; X is NH; Y is CH.sub.2; m is an integer of from 0 to 2; n is 0 or 1; and p is 0.

18. The method according to claim 1, wherein the compound of formula (I) is selected from N.sup.2-(3,4-dimethylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; N.sup.2-(3-chloro-4-methylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; 6-((4-phenylpiperazin-1-yl)methyl)-N.sup.2-(p-tolyl)-1,3,5-triazine-2,4-diamine; N.sup.2-phenyl-6-((4-phenylpiperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; and 6-((4-(4-fluorophenyl)piperazin-1-yl)methyl)-N.sup.2-phenyl-1,3,5-triazine-2,4-diamine; or a pharmaceutically acceptable salt thereof.

19. The method according to claim 1, wherein the disorder or condition is selected from diabetes, stroke and lung fibrosis.

20. A pharmaceutical composition comprising a compound selected from N.sup.2-(3,4-dimethylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; N.sup.2-(3-chloro-4-methylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; 6-((4-phenylpiperazin-1-yl)methyl)-N.sup.2-(p-tolyl)-1,3,5-triazine-2,4-diamine; N.sup.2-phenyl-6-((4-phenylpiperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; and 6-((4-(4-fluorophenyl)piperazin-1-yl)methyl)-N.sup.2-phenyl-1,3,5-triazine-2,4-diamine; or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient.

21. The method according to claim 1, wherein the disorder or condition is diabetes.

22. The method according to claim 1, wherein the disorder or condition is stroke.

23. The method according to claim 1, wherein the disorder or condition is lung fibrosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows dose-response curves for 4 different compounds of the invention in Nox4-transfected TRex-293 cells, at 11 concentrations obtained by serial dilution 1:3 of a 200 M solution of tested compound: A) N.sup.2-(3,4-dimethylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine, B) N.sup.2-(3-chloro-4-methylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine, C) 6-((4-phenylpiperazin-1-yl)methyl)-N.sup.2-(p-tolyl)-1,3,5-triazine-2,4-diamine, D) N.sup.2-phenyl-6-((4-phenylpiperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine, and E) 6-((4-(4-fluorophenyl)piperazin-1-yl)methyl)-N.sup.2-phenyl-1,3,5-triazine-2,4-diamine.

(2) FIG. 2 is a bar chart showing the mean infarct volume, in mm.sup.3, in brain from stroke induced mice treated by i.p. injection of N.sup.2-(3,4-dimethylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine (M4) or of vehicle (Control), respectively.

DETAILED DESCRIPTION OF THE INVENTION

(3) In general any term used herein shall be given its normal meaning as accepted within the field to which the present invention belongs. For the sake of clarity, however, some definitions will be given herein below, and shall apply throughout the specification and the appended claims, unless otherwise specified or apparent from the context.

(4) The term endocrine disorder refers to disorders of the endocrine system and may be as well endocrine gland hyposecretion as hypersecretion, or tumors of endocrine glands. Diabetes and polycystic ovarian syndrome are examples of endocrine disorders.

(5) The term cardiovascular disorder or disease comprises atherosclerosis, especially diseases or disorders associated with endothelial dysfunction including but not limited to hypertension, cardiovascular complications of Type I or Type II diabetes, intimal hyperplasia, coronary heart disease, cerebral, coronary or arterial vasospasm, endothelial dysfunction, heart failure including congestive heart failure, peripheral artery disease, restenosis, trauma caused by a stent, stroke, ischemic attack, vascular complications such as after organ transplantation, myocardial infarction, hypertension, formation of atherosclerotic plaques, platelet aggregation, angina pectoris, aneurysm, aortic dissection, ischemic heart disease, cardiac hypertrophy, pulmonary embolus, thrombotic events including deep vein thrombosis, injury caused after ischemia by restoration of blood flow or oxygen delivery as in organ transplantation, open heart surgery, angioplasty, hemorrhagic shock, angioplasty of ischemic organs including heart, brain, liver, kidney, retina and bowel.

(6) The term respiratory disorder or disease comprises bronchial asthma, bronchitis, allergic rhinitis, adult respiratory syndrome, cystic fibrosis, lung viral infection (influenza), pulmonary hypertension, idiopathic pulmonary fibrosis and chronic obstructive pulmonary diseases (COPD).

(7) The term allergic disorder includes hay fever and asthma.

(8) The term traumatism includes polytraumatism.

(9) The term disease or disorder affecting the metabolism includes obesity, metabolic syndrome and Type II diabetes.

(10) The term skin disease or disorder includes psoriasis, eczema, dermatitis, wound healing and scar formation.

(11) The term bone disorder includes osteoporosis, osteoporasis, osteosclerosis, periodontitis, and hyperparathyroidism.

(12) The term neurodegenerative disease or disorder comprises a disease or a state characterized by a central nervous system (CNS) degeneration or alteration, especially at the level of the neurons such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and muscular dystrophy. It further comprises neuro-inflammatory and demyelinating states or diseases such as leukoencephalopathies, and leukodystrophies.

(13) The term demyelinating is referring to a state or a disease of the CNS comprising the degradation of the myelin around the axons. In the context of the invention, the term demyelinating disease is intended to comprise conditions which comprise a process that demyelinate cells such as multiple sclerosis, progressive multifocal leukoencephalopathy (PML), myelopathies, any neuroinflammatory condition involving autoreactive leukocyte within the CNS, congenital metabolic disorder, a neuropathy with abnormal myelination, drug induced demyelination, radiation induced demyelination, a hereditary demyelinating condition, a prion induced demyelinating condition, encephalitis induced demyelination or a spinal cord injury. Preferably, the condition is multiple sclerosis.

(14) The term kidney disease or disorder includes diabetic nephropathy, renal failure, glomerulonephritis, nephrotoxicity of aminoglycosides and platinum compounds and hyperactive bladder. In a particular embodiment, the term according to the invention includes chronic kidney diseases or disorders.

(15) The term reproduction disorder or disease includes erectile dysfunction, fertility disorders, prostatic hypertrophy and benign prostatic hypertrophy.

(16) The term disease or disorder affecting the eye and/or the lens includes cataract including diabetic cataract, re-opacification of the lens post cataract surgery, diabetic and other forms of retinopathy.

(17) The term conditions affecting the inner ear includes presbyacusis, tinnitus, Meniere's disease and other balance problems, utriculolithiasis, vestibular migraine, and noise induced hearing loss and drug induced hearing loss (ototoxicity).

(18) The term inflammatory disorder or disease means inflammatory bowel disease, sepsis, septic shock, adult respiratory distress syndrome, pancreatitis, shock induced by trauma, bronchial asthma, allergic rhinitis, rheumatoid arthritis, chronic rheumatoid arthritis, arteriosclerosis, intracerebral hemorrhage, cerebral infarction, heart failure, myocardial infarction, psoriasis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, myelitis, ankylosing spondylitis, Reuter syndrome, psoriatic arthritis, spondylarthritis, juvenile arthritis or juvenile ankylosing spondylitis, reactive arthritis, infectious arthritis or arthritis after infection, gonococcal arthritis, syphilitic arthritis, Lyme disease, arthritis induced by angiitis syndrome, polyarteritis nodosa, anaphylactic angiitis, Luegenec granulomatosis, rheumatoid polymyalgia, articular cell rheumatism, calcium crystal deposition arthritis, pseudogout, non-arthritic rheumatism, bursitis, tendosynovitis, epicondyle inflammation (tennis elbow), carpal tunnel syndrome, disorders by repetitive use (typing), mixed form of arthritis, neuropathic arthropathy, hemorrhagic arthritis, vascular peliosis, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis induced by specific diseases, blood pigmentation, sickle cell disease and other hemoglobin abnormality, hyperlipoproteinemia, dysgammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Bechet's disease, systemic autoimmune disease erythematosus, multiple sclerosis and Crohn's disease or diseases like relapsing polychondritis, chronic inflammatory bowel diseases (IBD) or the related diseases which require the administration to a mammal in a therapeutic effective dose of a compound expressed by Formula (I) in a sufficient dose to inhibit NADPH oxidase.

(19) The term liver diseases or disorders include liver fibrosis, alcohol induced fibrosis, steatosis and non-alcoholic steatohepatitis.

(20) The term arthritis means acute rheumatic arthritis, chronic rheumatoid arthritis, chlamydial arthritis, chronic absorptive arthritis, anchylous arthritis, arthritis based on bowel disease, filarial arthritis, gonorrheal arthritis, gouty arthritis, hemophilic arthritis, hypertrophic arthritis, juvenile chronic arthritis, Lyme arthritis, neonatal foal arthritis, nodular arthritis, ochronotic arthritis, psoriatic arthritis or suppurative arthritis, or the related diseases which require the administration to a mammal in a therapeutic effective dose of a compound expressed by Formula (I) in a sufficient dose to inhibit NADPH oxidase.

(21) The term pain includes hyperalgesia associated with inflammatory pain.

(22) The term cancer means carcinoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelium sarcoma, lymphangiosarcoma, lymphangioendothelioma, periosteoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, renal cancer, prostatic carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, orchioncus, lung cancer, small-cell lung cancer, lung adenocarcinoma, bladder cancer or epithelial cancer) or the related diseases which require the administration to a mammal in a therapeutic effective dose of a compound expressed by the Formula (I) in a sufficient dose to inhibit NADPH oxidase.

(23) The term disease or disorders of the gastrointestinal system, includes gastric mucosa disorders ischemic bowel disease management, enteritis/colitis, cancer chemotherapy, or neutropenia.

(24) The term angiogenesis includes sprouting angiogenesis, intussusceptive angiogenesis, vasculogenesis, arteriogenesis and lymphangiogenesis. Angiogenesis is the formation of new blood vessels from pre-existing capillaries or post-capillary venules and occurs in pathological conditions such as cancers, arthritis and inflammation. A large variety of tissues, or organs comprised of organized tissues, can support angiogenesis in disease conditions including skin, muscle, gut, connective tissue, joints, bones and the like tissue in which blood vessels can invade upon angiogenic stimuli. As used herein, the term angiogenesis-dependent condition is intended to mean a condition where the process of angiogenesis or vasculogenesis sustains or augments a pathological condition. Vasculogenesis results from the formation of new blood vessels arising from angioblasts which are endothelial cell precursors. Both processes result in new blood vessel formation and are included in the meaning of the term angiogenesis-dependent conditions. Similarly, the term angiogenesis as used herein is intended to include de novo formation of vessels such as those arising from vasculogenesis as well as those arising from branching and sprouting of existing vessels, capillaries and venules.

(25) The term angiogenesis inhibitory, means which is effective in the decrease in the extent, amount, or rate of neovascularization. Effecting a decrease in the extent, amount, or rate of endothelial cell proliferation or migration in the tissue is a specific example of inhibiting angiogenesis. Angiogenesis inhibitory activity is particularly useful in the treatment of any cancers as it targets tumor growth process and in the absence of neovascularization of tumor tissue, the tumor tissue does not obtain the required nutrients, slows in growth, ceases additional growth, regresses and ultimately becomes necrotic resulting in killing of the tumor. Further, an angiogenesis inhibitory activity is particularly useful in the treatment of any cancers as it is particularly effective against the formation of metastases because their formation also requires vascularization of a primary tumor so that the metastatic cancer cells can exit the primary tumor and their establishment in a secondary site requires neovascularization to support growth of the metastases.

(26) As used herein, treatment and treating and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term treatment as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions.

(27) The term subject as used herein refers to mammals. For examples, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses and the like.

(28) An effective amount refers to an amount of a compound that confers a therapeutic effect on the treated subject. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).

(29) The term inhibitor used in the context of the invention is defined as a molecule that inhibits completely or partially the activity of Nox, in particular Nox4, and/or inhibits or reduces the generation of reactive oxygen species (ROS).

(30) Pharmaceutically acceptable means being useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes being useful for veterinary use as well as human pharmaceutical use.

(31) The term Cn, where n is an integer, specifies that a radical or moiety contains n carbon atoms. The term CnCm, where m and n are both integers, and m>n, refers to a radical or moiety containing n, n+1, n+2, . . . or m carbon atoms. Thus, the term C1-C6 alkyl refers to an alkyl radical that may contain 1, 2, 3, 4, 5 or 6 carbon atoms. The term C0 alkyl refers to a covalent bond.

(32) An alkyl moiety according to the invention may be branched or linear, e.g. selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

(33) A C1-C6 alkyl according to the invention more particularly may be selected from C1-05 alkyl, e.g. from C1-C4 alkyl, from C1-C3 alkyl, from C1-C2 alkyl, or may be methyl.

(34) The term C2-C6 alkenyl refers to a straight or branched chain alkenyl having from 2 to 6 carbon atoms in the chain and that may have any available number of double bonds in any available positions. The configuration of the double bond may be (E) or (Z). Examples are vinyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, and 5-hexenyl. A C2-C6 alkenyl according to the invention, more specifically may be a C2-C4 alkenyl, or a C2-C3 alkenyl.

(35) The term C2-C6 alkynyl refers to a straight or branched chain alkynyl having from 2 to 6 carbon atoms in the chain and that may have any available number of triple bonds in any available positions. Examples are ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, and 2-pentene-4-ynyl. A C2-C6 alkynyl according to the invention, more specifically may be a C2-C4 alkynyl, or a C2-C3 alkynyl.

(36) The term C3-C6 cycloalkyl refers to a cyclic alkyl radical having from 3 to 6 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

(37) By substituted with at least one halogen is meant that at least one hydrogen is replaced by a halogen, e.g. F. An example of an alkyl substituted with at least one halogen is trifluoromethyl.

(38) As used herein, and unless otherwise specified, the term halogen (or halo) means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Any halogen according to the invention more particularly may be selected from F and Cl.

(39) In a compound of formula (I) as provided herein, each R.sup.1, R.sup.2 and R.sup.3 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(40) In some embodiments, each R.sup.1, R.sup.2 and R.sup.3 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, and C1-C6 alkyl, said alkyl, optionally being substituted with at least one halogen.

(41) In some other embodiments, each R.sup.1, R.sup.2 and R.sup.3 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, and C1-C6 alkyl, said alkyl, optionally being substituted with at least one halogen.

(42) As noted herein above, each R.sup.1 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(43) In some embodiments, each R.sup.1 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(44) In some other embodiments, each R.sup.1 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(45) In still other embodiments, each R.sup.1 is independently selected from halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(46) In some particular embodiments, each R.sup.1 is independently selected from halogen and C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(47) In some embodiments, each R.sup.1 is independently selected from C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(48) In some embodiments, each R.sup.1 is independently selected from halogen.

(49) In any of the above embodiments, when R.sup.1 is halogen, it especially may be selected from F and Cl, and in particular R.sup.1 may be Cl.

(50) As noted herein above, each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(51) In some embodiments, each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(52) In some other embodiments, each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(53) In still other embodiments, each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, and C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(54) In further embodiments, each R.sup.2 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, and R.sup.5S(CH.sub.2).sub.q.

(55) In some embodiments, each R.sup.2 is independently selected from R.sup.5O(CH.sub.2).sub.q, and R.sup.5S(CH.sub.2).sub.q.

(56) In some other embodiments, each R.sup.2 is independently selected from halogen, and R.sup.5O(CH.sub.2).sub.q.

(57) In some embodiments, each R.sup.2 is independently selected from R.sup.5O(CH.sub.2).sub.q.

(58) In some other embodiments, each R.sup.2 is independently selected from halogen.

(59) As noted herein above, R.sup.3 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(60) In some embodiments, R.sup.3 is independently selected from halogen, R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, CN(CH.sub.2).sub.q, C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(61) In some other embodiments, R.sup.3 is R.sup.6R.sup.7N(CH.sub.2).sub.q.

(62) In a compound of formula (I), each R.sup.4 is independently selected from halogen, e.g. F, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(63) In some embodiments, each R.sup.4 is independently selected from halogen, e.g. F and C1-C6 alkyl, said alkyl, optionally being substituted with at least one halogen.

(64) In a compound of formula (I), any R.sup.5, when present, is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(65) In some embodiments, any R.sup.5, when present, is independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(66) In some embodiments, any R.sup.5, when present, is independently selected from H and C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(67) In some embodiments, any R.sup.5, when present, is independently selected from C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(68) In a compound of formula (I), each R.sup.6 and R.sup.7 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; said alkyl, alkenyl, alkynyl and cycloalkyl optionally being substituted with at least one halogen.

(69) In some embodiments, each R.sup.6 and R.sup.7 is independently selected from H and C1-C6 alkyl, said alkyl optionally being substituted with at least one halogen.

(70) In some embodiments, R.sup.6 is as defined herein above and R.sup.7 is H. In other embodiments, both R.sup.6 and R.sup.7 are H.

(71) In a compound of formula (I) as defined herein, X is NH, CH.sub.2 or C(O). In some embodiments, X is NH or CH.sub.2. In other embodiments, X is NH.

(72) In a compound of formula (I) as defined herein, Y is NH, CH.sub.2 or C(O). In some embodiments, Y is NH or CH.sub.2. In other embodiments, Y is CH.sub.2.

(73) The integer m, representing the number of moieties R.sup.1 in a compound of formula (I), ranges from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. In some embodiments, m is at least 1, e.g. m is 1-5, 1-4, or 1-3. In some embodiments, m is 1 or 2, e.g. m is 2.

(74) In some embodiments, when m is an integer of 1 or higher, one R.sup.1 is attached to the phenyl ring in para position or in meta position. In other words, the phenyl ring is substituted on any of carbon atoms number 3, 4 or 5 of the phenyl ring, assuming that the X link to the triazine ring is attached at carbon atom number 1 of the phenyl ring.

(75) In some embodiments, when m is an integer of 1 or higher, one R.sup.1 is attached to the phenyl ring in para position.

(76) In some embodiments, when m is an integer of 2 or higher, one R.sup.1 is attached to the phenyl ring in para position and one R.sup.1 is attached in meta position.

(77) The integer n, representing the number of moieties R.sup.2 in a compound of formula (I), ranges from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. In some embodiments, n is at least 1, e.g. n is 1-5, 1-4, or 1-3. In some embodiments, n is 1 or 2, e.g. n is 1. In some embodiments, n is 0 or 1.

(78) In some embodiments, when n is an integer of 1 or higher, one R.sup.2 is attached to the phenyl ring in para position or in meta position. In other words, the phenyl ring is substituted on any of carbon atoms number 3, 4 or 5 of the phenyl ring, assuming that the Y link to the triazine ring is attached at carbon atom number 1 of the phenyl ring.

(79) In some embodiments, when n is an integer of 1 or higher, e.g. n is 1, one R.sup.2 is attached to the phenyl ring in meta position, e.g. R.sup.2 is R.sup.5O(CH.sub.2).sub.q in meta position. For example, n is 1 and R.sup.2 is in meta position and is R.sup.5O(CH.sub.2).sub.q.

(80) In some embodiments, when n is an integer of 1 or higher, e.g. n is 1, one R.sup.2 is attached to the phenyl ring in para position, e.g. R.sup.2 is a halogen, such as F, in para position.

(81) The integer p, representing the number of moieties R.sup.4 in a compound of formula (I), ranges from 0 to 4, from 0 to 3, or from 0 to 2, e.g. p is 0 or 1. In some embodiments, p is 0.

(82) The integer q in any moiety R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, and CN(CH.sub.2).sub.q is selected from 0, 1, 2 and 3. In some embodiments, any q is selected from 0, 1 and 2; or from 0 and 1. In some embodiments, q is 0, i.e. any moiety R.sup.5O(CH.sub.2).sub.q, R.sup.5S(CH.sub.2).sub.q, R.sup.6R.sup.7N(CH.sub.2).sub.q, or CN(CH.sub.2).sub.q is R.sup.5O, R.sup.5S, R.sup.6R.sup.7N, or CN.

(83) In some particular embodiments, X is NH and Y is CH.sub.2. In some other particular embodiments, X is NH, Y is CH.sub.2, and p is 0. In still other particular embodiments, X is NH, Y is CH.sub.2, p is 0, and R.sup.3 is R.sup.6R.sup.7N.

(84) Many further embodiments are possible and contemplated within the scope of formula (I), some of which are illustrated in a non-limiting fashion in the following Table 1.

(85) TABLE-US-00001 TABLE 1 Exemplary embodiments of a compound of formula (I) according to the invention Special feature(s) of embodiment Strucural formula of embodiment (a) X is NH (Ia) (b) Y is CH.sub.2 (Ib) (C) R.sup.3 is NR.sup.6R.sup.7(CH.sub.2)q (Ic) (d) p is 0 0 (Id) (e) m is at least 1, and one R.sup.1 is in para position (Ie) (f) n is at least 1, and one R.sup.2 is in meta position (If) (g) R.sup.3 is NR.sup.6R.sup.7(CH.sub.2)q, and q is 0 (Ig) (h) = (a) + (b) (Ih) (i) = (h) + (c) (Ii) (j) = (i) + (d) (Ij) (k) = (h) + (g) (Ik) (m) = (k) + (d) (Im) (n) = (m) wherein R.sup.6 and R.sup.7 are both H (In)

(86) In Table 1 the sum of e.g. (a) and (b), written as (h)=(a)+(b), refers to an embodiment (embodiment (h)) comprising the features of embodiments (a) and (b), respectively, i.e. wherein X has been specified as NH, and Y has been specified as CH.sub.2, and so on with other embodiments.

(87) Unless apparent from the context or specified herein, any reference to a compound of formula (I) also is intended as applying to a compound any one of the formulas (Ia) to (In), as illustrated in Table 1. It should however be realized that many other embodiments are also possible within the scope of the invention.

(88) In some particular embodiments, the compound of the invention is selected from N.sup.2-(3,4-dimethylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; N.sup.2-(3-chloro-4-methylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; 6-((4-phenylpiperazin-1-yl)methyl)-N.sup.2-(p-tolyl)-1,3,5-triazine-2,4-diamine; N.sup.2-phenyl-6-((4-phenylpiperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine; and 6-((4-(4-fluorophenyl)piperazin-1-yl)methyl)-N.sup.2-phenyl-1,3,5-triazine-2,4-diamine; or from pharmaceutically acceptable salts thereof.

(89) The compounds of formula (I) can be prepared by methods well known in the art, from readily available starting materials using general methods and procedures. Some compounds of formula (I) may be commercially available, e.g. from Vitas laboratories, Moscow, 125252, Russia.

(90) The compounds of the present invention are Nox inhibitors. More specifically, the compounds of the present invention are Nox4 inhibitors. The capacity of inhibiting predominantly one particular Nox iso form, i.e. Nox4, is considered to be an important advantage of the present compounds, in view of the fact that Nox isoforms not only are involved in diseases, as Nox4, but also have various important biological functions in the living body.

(91) Depending on the process conditions the end products of formula (I) are obtained either in neutral or salt form. Both the free base and the free acid, as well as the salts of these end products are within the scope of the invention. Acid addition salts of the inventive compounds may in a manner known per se be transformed into the free base using basic agents such as alkali or by ion exchange. The free base obtained may also form salts with organic or inorganic acids. Alkali addition salts of the inventive compounds may in a manner known per se be transformed into the free acid by using acidic agents such as acid or by ion exchange. The free acid obtained may also form salts with organic or inorganic bases.

(92) In the preparation of acid or base addition salts, preferably such acids or bases are used which form suitably therapeutically acceptable salts. Examples of such acids are hydrohalogen acids, sulfuric acid, phosphoric acid, nitric acid, aliphatic, alicyclic, aromatic or heterocyclic carboxylic or sulfonic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, pyruvic acid, p-hydroxybenzoic acid, embonic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, halogenbenzenesulfonic acid, toluenesulfonic acid or naphthalenesulfonic acid. Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases such as alkoxides, alkyl amides, alkyl and aryl amines, and the like. Examples of bases useful in preparing salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

(93) There may be several stereoisomers of the compounds of the invention, including enantiomers and diastereomers. Enantiomers can be present in their pure forms, or as racemic (equal) or unequal mixtures of two enantiomers. Diastereomers can be present in their pure forms, or as mixtures of diastereomers. Diastereomers also include geometric isomers, which can be present in their pure cis or trans forms or as mixtures of those.

(94) Pharmaceutical formulations are usually prepared by mixing the active substance, i.e. a compound of the invention, or a pharmaceutically acceptable salt thereof, with conventional pharmaceutical excipients. The formulations can be further prepared by known methods such as granulation, compression, microencapsulation, spray coating, etc. The formulations may be prepared by conventional methods in the dosage form of tablets, capsules, granules, powders, syrups, suspensions, suppositories or injections. Liquid formulations may be prepared by dissolving or suspending the active substance in water or other suitable vehicles. Tablets and granules may be coated in a conventional manner.

(95) For clinical use, the compounds of the invention are formulated into pharmaceutical formulations for oral, rectal, parenteral or other mode of administration. These pharmaceutical preparations are a further object of the invention.

(96) Usually the effective amount of active compounds is between 0.1-95% by weight of the preparation, preferably between 0.2-20% by weight in preparations for parenteral use and preferably between 1 and 50% by weight in preparations for oral administration.

(97) The dose level and frequency of dosage of the specific compound will vary depending on a variety of factors including the potency of the specific compound employed, the metabolic stability and length of action of that compound, the patient's age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the condition to be treated, and the patient undergoing therapy. The daily dosage may, for example, range from about 0.001 mg to about 100 mg per kilo of body weight, administered singly or multiply in doses, e.g. from about 0.01 mg to about 25 mg each. Normally, such a dosage is given orally but parenteral administration may also be chosen.

(98) In the preparation of pharmaceutical formulations containing a compound of the present invention in the form of dosage units for oral administration the compound selected may be mixed with solid, powdered ingredients, such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, or another suitable ingredient, as well as with disintegrating agents and lubricating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes. The mixture is then processed into granules or pressed into tablets.

(99) Soft gelatine capsules may be prepared with capsules containing a mixture of the active compound or compounds of the invention, vegetable oil, fat, or other suitable vehicle for soft gelatine capsules. Hard gelatine capsules may contain granules of the active compound. Hard gelatine capsules may also contain the active compound in combination with solid powdered ingredients such as lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives or gelatine.

(100) Dosage units for rectal administration may be prepared (i) in the form of suppositories which contain the active substance mixed with a neutral fat base; (ii) in the form of a gelatine rectal capsule which contains the active substance in a mixture with a vegetable oil, paraffin oil or other suitable vehicle for gelatine rectal capsules; (iii) in the form of a ready-made micro enema; or (iv) in the form of a dry micro enema formulation to be reconstituted in a suitable solvent just prior to administration.

(101) Liquid preparations for oral administration may be prepared in the form of syrups or suspensions, e.g. solutions or suspensions containing from 0.2% to 20% by weight of the active ingredient and the remainder consisting of sugar or sugar alcohols and a mixture of ethanol, water, glycerol, propylene glycol and polyethylene glycol. If desired, such liquid preparations may contain colouring agents, flavouring agents, saccharine and carboxymethyl cellulose or other thickening agent. Liquid preparations for oral administration may also be prepared in the form of a dry powder to be reconstituted with a suitable solvent prior to use.

(102) Solutions for parenteral, e.g. intravenous, administration may be prepared as a solution of a compound of the invention in a pharmaceutically acceptable solvent, preferably in a concentration from 0.1% to 10% by weight. These solutions may also contain stabilizing ingredients and/or buffering ingredients and are dispensed into unit doses in the form of ampoules or vials. Solutions for parenteral administration may also be prepared as a dry preparation to be reconstituted with a suitable solvent extemporaneously before use. The compounds of the present invention may also be used or administered in combination with one or more additional therapeutically active agents. The components may be in the same formulation or in separate formulations for administration simultaneously or sequentially.

(103) Accordingly, in a further aspect of the invention, there is provided a combination product comprising:

(104) (A) a compound of the invention, as defined herein; and

(105) (B) another therapeutic agent; whereby (A) and (B) is formulated in admixture with a pharmaceutically acceptable excipient.

(106) Such combination products provide for the administration of a compound of the invention in conjunction with the other therapeutic agent, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises a compound of the invention, and at least one comprises the other therapeutic agent, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including a compound of the invention and the other therapeutic agent).

(107) Thus, there is further provided:

(108) (1) a pharmaceutical formulation including a compound of the invention, as hereinbefore defined, another therapeutic agent, and a pharmaceutically acceptable excipient, e.g. an adjuvant, diluent or carrier; or

(109) (2) a kit of parts comprising, as components:

(110) (a) a pharmaceutical formulation including a compound of the invention, as defined herein, in admixture with a pharmaceutically acceptable excipient, e.g. an adjuvant, diluent or carrier; and

(111) (b) a pharmaceutical formulation including another therapeutic agent in admixture with a pharmaceutically acceptable excipient, e.g. an adjuvant, diluent or carrier, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

(112) In some particular embodiments, the compound of the invention is used in a combination with an antitumor agent in the treatment of a malignant hyperproliferative disease. Such combination therapy may be particularly useful in cancer chemotherapy, to counteract an anti-apoptotic effect of Nox4 that may lead to tumor resistance to the antitumor agent.

(113) Thus, there is further provided:

(114) (1) a pharmaceutical formulation including a compound of the invention, as hereinbefore defined, an antitumor agent, and a pharmaceutically acceptable excipient, e.g. an adjuvant, diluent or carrier; or

(115) (2) a kit of parts comprising, as components:

(116) (a) a pharmaceutical formulation including a compound of the invention, as defined herein, in admixture with a pharmaceutically acceptable excipient, e.g. an adjuvant, diluent or carrier; and

(117) (b) a pharmaceutical formulation including an antitumor agent in admixture with a pharmaceutically acceptable excipient, e.g. an adjuvant, diluent or carrier, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

(118) The components (a) and (b) in any of the above kit of parts may be administered at the same time, in sequence, or separately from each other.

(119) The compounds of the present invention may also be used or administered in combination with other modes of treatment such as irradiation for the treatment of cancer.

(120) According to one aspect, there is provided a method of inhibiting the activity of Nox, in particular Nox4, in a patient in need thereof, by administering to said patient a therapeutically effective amount of a compound of formula (I) as defined herein. The patient may be any mammal, but preferably is a human.

(121) The patient to be treated may be one suffering from a condition or disorder associated with an elevated activity of Nox, in particular Nox4, or a patient at risk of developing such a condition or disorder. Examples of such conditions and disorders are cardiovascular disorders, respiratory disorders, metabolism disorders, skin disorders, bone disorders, neuroinflammatory and/or neurodegenerative disorders, kidney diseases, reproduction disorders, diseases affecting the eye and/or the lens and/or conditions affecting the inner ear, inflammatory disorders, liver diseases, pain, cancers, allergic disorders, traumatisms, septic, hemorrhagic and anaphylactic shock, diseases or disorders of the gastrointestinal system, angiogenesis, angiogenesis-dependent conditions, lung infections, acute lung injury, pulmonary arterial hypertension, obstructive lung disorders, fibrotic lung disease, and lung cancer.

(122) In one embodiment, the compounds of the present invention are for use in the treatment of stroke. In one particular embodiment, the stroke is ischemic. The compounds of the present invention are considered to have neuroprotective activity in the treatment of stroke. Therefore, the compounds of the present invention suitable are used in combination with removal of blood clots in the treatment of ischemic stroke. In one particular embodiment, the compounds of the present invention are used in combination with tPA in the treatment of ischemic stroke.

(123) The invention will be illustrated by the following, non-limiting Examples.

EXAMPLES

Example 1

Cell-Based Assays and Analytical Chemistry

(124) 1. Cell Viability

(125) 1.1 Celltiter-Blue Cell Viability Assay (Promega)

(126) The assay is based on the ability of the cells to reduce resazurin to resorufin as a measure of viability. TREx-293 Nox4 cells were cultured in a T-225 flask, collected by trypsination and re-suspended in cell medium. 20,000 cells in 90 l were seeded to 96-well cell culture plates (black with transparent bottom). One background plate with 90 l cell medium only was also prepared.

(127) After 24 hours, 10 l of compound, diluted to 10 times final concentration in 37 C. cell medium, were added to cell and background plates. The compounds were tested in duplicate at a final concentration of 10 M. Chlorpromazine, at a final concentration of 100 M, was added as positive control. After 24 hours of treatment, 20 l of CellTiter-Blue reagent were added and the plate was incubated for 120 min at 37 C. Resorufin fluorescence was read in Victor2V plate reader. All experimental values were corrected for background before analysis of the cell viability.

(128) 1.2 CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega)

(129) The assay is based on lactate dehydrogenase (LDH) activity in surrounding cell medium as a measure of membrane integrity. Membrane integrity can be affected by apoptosis, necrosis or chemicals. TREx-293 Nox4 cells were cultured in a T-225 flask, collected by trypsination and re-suspended in HBSS to 100,000 cells per ml. 90 l of cell suspension were added to each well of a V-bottom polypropylene 96-well plate. One background plate was prepared with HBSS only. Compounds were diluted in HBSS to 10 times final concentration and 10 l was added per well. The compounds were tested in duplicate at a final concentration of 10 M.

(130) Plates were incubated 3 hours at 37 C. 45 minutes before end of incubation time, 10 l of lysis solution (Triton X-100) were added to total control wells to estimate total LDH content of cells. Spontaneous LDH leakage was determined with un-treated cells.

(131) Cell plates were centrifuged 250g for 5 minutes and 50 l of supernatant were transferred to 96-well Spectraplates. 50 l of reconstituted substrate mix were added and plates were incubated for 30 minutes at room temperature. 50 l of stop solution were added and plates were read in SpectraMax at a wavelength of 490 nm. Compound specific background was subtracted and % cytotoxcity was calculated as:
[(ExperimentalSpontaneous)/(TotalSpontaneous)]*100%.

(132) When tested in the two cell viability assays, none of the inventive compounds showed any significant cell toxicity effects.

(133) 2. Dose-Response Curves

(134) Dose-response measurements with the Amplex Red based assay were performed as follows: Compound serial dilution was carried out using the system based on the liquid handler Janus (Perkin Elmer) and scheduling software Overlord (Process Analysis and Automation).

(135) Starting with compound plates with 15 l 10 mM compound stock solution in DMSO, 10 l of DMSO were added to columns of compound plate (Flexdrop). Serial dilution was performed by adding 5 l compound solution to 10 l DMSO (1:3) to 11 concentrations. To each well of the compound plate 90 l of assay buffer were added. After mixing, 10 l were transferred from each well of the compound plate to wells of an assay plate, followed by addition of 20 l detection mix and 20 l of a suspension of TREx-293 Nox4 cells. The assay plate then was incubated for 40-60 min at room temperature.

(136) Data was analyzed using a custom calculation template in Activitybase XE (IDBS). Raw fluorescence data was transformed to % inhibition using the built-in formula:

(137) $\%\mathrm{inhibition}=100-\frac{{\mathrm{RawData}}_{\mathrm{Compound}}-{\mathrm{RawData}}_{\mathrm{Low}}}{{\mathrm{RawData}}_{\mathrm{High}}-{\mathrm{RawData}}_{\mathrm{Low}}}100$

(138) Dose-response curves were fitted using non-linear regression with four parameter logistic formula. FIGS. 1A-E show dose-response curves for some compounds of the invention. The tested compounds have 1050 values ranging from about 1 M to about 68 M.

Example 2

In Vivo Study: Stroke Animal Model

(139) Mice (10-12 wk), wild type C57BI6, 6 animals, were used:

(140) Control (n=3) stroke induced mouse treated i.p. with vehicle 2 h and 12 h.

(141) Test (n=3) stroke induced mouse treated i.p. with inventive compound M4 (N.sup.2-(3,4-dimethylphenyl)-6-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-1,3,5-triazine-2,4-diamine).

(142) M4 preparation for intra peritoneal i.p. administration: 3.5 mg of M4 were first dissolved in 1 ml of DMSO. After the substance was totally dissolved, 5 ml of 20% Cremophor ELP in PBS (phosphate buffered saline solution) was added. Thereafter an additional 4 ml of PBS was added. The 20% Cremophor ELP solution was prepared by dissolving 10.5 g of Cremophor in 50 ml of PBS.

(143) Final concentration of the injection solution: 0.35 mg/ml.

(144) Injection volume of M4: The dose was 3.2 mg/kg, calculated for each animal at each event of administration (2 h and 12 h).

(145) Procedure:

(146) 1. Transient middle cerebral artery occlusion (tMCAO) by blocking with a filament for 1 h. 2. Reperfusion after 1 h by removing filament 3. Injection of M4 by i.p. after 2 h and repeated injection i.p. after 12 h 4. Sacrifice the animal after 24 h and infarct size is determined by triphenyl tetrazolium chloride (TTC) stainingThus the procedure of middle cerebral artery occlusion (MCAO); 1 h occlusion and 23 h reperfusion. 5. Stroke analysis was performed as described previously (Experimental Neurology 200 (2006), pp. 480-485; Circulation May 1, 2007, pp. 2323-2330). To determine infarct size, mice were killed 24 h after tMCAO, pMCAO, or cortical photothrombosis. Brains were cut in 2-mm-thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained with 2% TTC (Sigma-Aldrich) to visualize the infarcts. Planimetric measurements (ImageJ software, United States National Institutes of Health), calculating lesion volumes, were corrected for brain edema as described previously (Ann Neurol 2003; 54:330-342).
Animal Studies

(147) Animal studies were approved by the Regierung von Unterfranken and conducted according to the recently published recommendations for research in mechanism-driven basic stroke studies. Adult male C57/BL6 mice (20 to 25 g) were purchased from Charles River (Sulzfeld, Germany). The tMCAO model was used to induce focal cerebral ischemia as described in detail elsewhere. Briefly, mice were anesthetized with 2% isoflurane in a 70% N2O/30% O2 mixture. A servo-controlled heating blanket was used to maintain core body temperature close to 37 C. throughout surgery. After a midline neck incision was made, a standardized silicon rubber-coated 6.0 nylon monofilament (60-1720RE, Doccol, Redlands, Calif.) was inserted into the right common carotid artery and advanced via the internal carotid artery to occlude the origin of the MCA. After 1 hour, mice were reanesthetized, and the occluding filament was removed to allow reperfusion. All animals were operated on by the same operator to reduce infarct variability; operation time per animal did not exceed 15 minutes.

(148) Determination of Infarct Size

(149) Mice were killed 24 hours after tMCAO. Brains were quickly removed and cut into 2-mm-thick coronal sections using a mouse brain slice matrix. The slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, Seelze, Germany) in PBS to visualize the infarctions. Planimetric measurements (ImageJ software, National Institutes of Health, Bethesda, Md.) were performed by researchers blinded to the treatment group and were used to calculate lesion volumes, which were corrected for brain edema as described elsewhere. The occurrence of Intra Cerebral Hemorrhage (ICH) was macroscopically assessed on whole brains and again after the 2-mm-thick coronal brain slices were cut (see above) before TTC staining. Brains showing ICH were excluded from the assessment of infarct volume. The results for the tree mice treated with M4 and the three control mice, respectively, are shown in Table 2. FIG. 2 is a bar chart showing the mean infarct volume for the group treated with M4 and the control group, respectively.

(150) TABLE-US-00002 TABLE 2 Infarct volume, mm.sup.3 Animals treated with M4 Control animals 73 107 145 224 134 204